Cloning and expression of a thermostable β-1,3-1,4-glucanase from Bacillus amyloliquefaciens ATCC 23350

We report the successful cloning and expression of a novel, heat-stable β-1,3-1,4-glucanase gene from Bacillus amyloliquefaciens ATCC 23350 which differs from the cloning of an enzyme previously isolated from this same bacterium. The enzymatic and specific activities of recombinant β-1,3-1,4-glucanase in high-cell-density fermentation were 80 U/ml and 91 U/mg, respectively. Following a three-step purification, the enzyme obtained was noted to be purified by 54.6-fold and had a molecular weight of approximately 26 kDa. The half-life of recombinant β-1,3-1,4-glucanase at 90°C was 21 min, which is markedly higher than those of the previously reported β-1,3-1,4-glucanases isolated from the same bacterium. The recombinant enzyme also exhibited maximal activity at pH 6.0 and 50°C as well as a strict preference for oat β-glucan and lichenan. The metal ions Cu2+ and Mn2+ at a 1 mM concentration significantly enhanced enzymatic activity; however, other metallic ions, such as Fe3+, Zn2+, Fe2+, and Li+, inhibited enzymatic activity. The K m and V max values estimated for oat β-glucan were 12 mg/ml and 7500 U/min per milligram of protein, respectively. These favorable properties make this enzyme a potential candidate for utilization in the brewing and animal-feed additives industries.